Effects of Exposure to Low Zearalenone Concentrations Close to the EU Recommended Value on Weaned Piglets’ Colon

Pigs are the most sensitive animal to zearalenone (ZEN) contamination, especially after weaning, with acute deleterious effects on different health parameters. Although recommendations not to exceed 100 µg/kg in piglets feed exists (2006/576/EC), there are no clear regulations concerning the maximum limit in feed for piglets, which means that more investigations are necessary to establish a guidance value. Due to these reasons, the present study aims to investigate if ZEN, at a concentration lower than the EC recommendation for piglets, might affect the microbiota or induce changes in SCFA synthesis and can trigger modifications of nutritional, physiological, and immunological markers in the colon (intestinal integrity through junction protein analysis and local immunity through IgA production). Consequently, the effect of two concentrations of zearalenone were tested, one below the limit recommended by the EC (75 µg/kg) and a higher one (290 µg/kg) for comparison reasons. Although exposure to contaminated feed with 75 µg ZEN/kg feed did not significantly affect the observed parameters, the 290 µg/kg feed altered several microbiota population abundances and the secretory IgA levels. The obtained results contribute to a better understanding of the adverse effects that ZEN can have in the colon of young pigs in a dose-dependent manner.


Introduction
Zearalenone (ZEN) is an estrogenic nonsteroidal mycotoxin which contaminates cereal grains and their derived products [1,2]. Multiple Fusarium species, including F. graminearum and F. culmorum produce ZEN that presents a significant risk for the livestock industry and an ongoing threat to human and animal health [3,4]. Sensitivity to ZEN varies according to species, swine being reported as the most sensitive among the livestock animals, 1.1 mg ZEN/kg feed for 18 days being enough to induce reproductive and endocrinal disorders in post-weaning gilts, affecting their ovarian follicles in a dose-related manner [5]. A survey of the last nine years reported by the Austrian Biomin Company shows that ZEN was detected in 40-71% of swine feed samples [6]. However, at the level of the European Commission, there are no regulations regarding the maximum limit allowed in the feed for young pigs, though only recommendations, meaning that additional studies are necessary to establish a precise regulation with this limit. For example, the effects of ZEN on the pig gut microbiota have not yet been sufficiently studied, especially at concentrations lower than 100 ppb [7]. A stable microbiome with well-balanced bacterial populations is necessary to maintain normal body function and prevent symptoms such as dysbiosis [8]. Generally, and especially in 2. Results

Effect of ZEN on the Percentage of Animals with Diarrhoea and on Animal Performances
During the experimental period, the piglets presenting diarrhoea symptoms were registered for each experimental group, and the results are presented in Figure 1. Exposure to zearalenone has increased the number of animals with diarrhoea, directly proportional to the concentration of ZEN (37.5% piglets with diarrhoea in the ZEN 75 group, respectively, 50% in the ZEN 290 group versus only 12.5% in the Control group).

Effect of ZEN on the Percentage of Animals with Diarrhoea and on Animal Performances
During the experimental period, the piglets presenting diarrhoea symptoms were registered for each experimental group, and the results are presented in Figure 1. Exposure to zearalenone has increased the number of animals with diarrhoea, directly proportional to the concentration of ZEN (37.5% piglets with diarrhoea in the ZEN 75 group, respectively, 50% in the ZEN 290 group versus only 12.5% in the Control group). Although slight differences between experimental groups could be observed for the growth parameters, these differences were not significant ( Table 1). The final body weight of piglets from the ZEN 75 and ZEN 290 group was lower compared to the Control. Slight decreases in ADG and ADFI could also be observed in ZEN experimental groups when compared with the Control group, but they were not significant. As such, ADG and ADFI parameters were similar for ZEN 75 and ZEN 290 (0.377 and 0.370 kg for ADG and 1.038 and 1.042 kg for ADFI) and were lower than the values in the Control group (0.424 kg and 1.144 kg). The best feed efficiency was observed for the Control group with a 2.6 ratio, compared to 2.77 for the ZEN 75 group and 2.82 for the ZEN 290 group; however, the differences were not significant.

Relative Abundance of Several Important Microbial Populations under the Influence of ZEN
Five bacterial genera were chosen as markers (Lactobacillus, Bifidobacterium, Prevotella, Enterobacter, and Clostridium) for a healthy intestinal tract based on the scientific literature and our previous studies. Their corresponding populations were determined from samples of colonic content ( Figure 2) and calculated as relative abundances. At 75 ppb, ZEN significantly influenced only the Lactobacillus population lowering it by 61% (p < 0.05, Figure 2), while not affecting Prevotella, Clostridium, Enterobacter, and Bifidobacterium when compared to control ( Figure 2). By contrast, Prevotella, Clostridium, and Enterobacter relative abundances were significantly impacted by the presence of ZEN at 290 ppb, registering an increase over the Control group and ZEN 75 group. The most spectacular change was observed for the Enterobacter population, increasing 50 times over that of the Control Although slight differences between experimental groups could be observed for the growth parameters, these differences were not significant ( Table 1). The final body weight of piglets from the ZEN 75 and ZEN 290 group was lower compared to the Control. Slight decreases in ADG and ADFI could also be observed in ZEN experimental groups when compared with the Control group, but they were not significant. As such, ADG and ADFI parameters were similar for ZEN 75 and ZEN 290 (0.377 and 0.370 kg for ADG and 1.038 and 1.042 kg for ADFI) and were lower than the values in the Control group (0.424 kg and 1.144 kg). The best feed efficiency was observed for the Control group with a 2.6 ratio, compared to 2.77 for the ZEN 75 group and 2.82 for the ZEN 290 group; however, the differences were not significant.

Relative Abundance of Several Important Microbial Populations under the Influence of ZEN
Five bacterial genera were chosen as markers (Lactobacillus, Bifidobacterium, Prevotella, Enterobacter, and Clostridium) for a healthy intestinal tract based on the scientific literature and our previous studies. Their corresponding populations were determined from samples of colonic content ( Figure 2) and calculated as relative abundances. At 75 ppb, ZEN significantly influenced only the Lactobacillus population lowering it by 61% (p < 0.05, Figure 2), while not affecting Prevotella, Clostridium, Enterobacter, and Bifidobacterium when compared to control ( Figure 2). By contrast, Prevotella, Clostridium, and Enterobacter relative abundances were significantly impacted by the presence of ZEN at 290 ppb, registering an increase over the Control group and ZEN 75 group. The most spectacular change was observed for the Enterobacter population, increasing 50 times over that of the Control group (p < 0.05) (Figure 2), followed by Clostridium, with an increase of 17.7 times (p < 0.05) and Prevotella with the lowest abundance increase, of only eight times over the Control group (p < 0.05) (Figure 2). Similar to the ZEN 75 group, ZEN at 290 ppb decreased the abundance of the Lactobacillus population by over 60% compared to the Control ( Figure 2). Bifidobacterium relative abundance was also significantly lowered, registering a decrease of 6.18% when compared with the Control group (p < 0.05) ( Figure 2). Similar to the ZEN 75 group, ZEN at 290 ppb significantly decreased the abundance of the Lactobacillus population by over 60% compared to the Control (Figure 2). In addition, Bifidobacterium relative abundance was also significantly lowered, registering a decrease of 6.18% when compared with the Control group (p < 0.05) ( Figure 2). group (p < 0.05) (Figure 2), followed by Clostridium, with an increase of 17.7 times (p < 0.05) and Prevotella with the lowest abundance increase, of only eight times over the Control group (p < 0.05) ( Figure 2). Similar to the ZEN 75 group, ZEN at 290 ppb decreased the abundance of the Lactobacillus population by over 60% compared to the Control ( Figure  2). Bifidobacterium relative abundance was also significantly lowered, registering a decrease of 6.18% when compared with the Control group (p < 0.05) ( Figure 2). Similar to the ZEN 75 group, ZEN at 290 ppb significantly decreased the abundance of the Lactobacillus population by over 60% compared to the Control ( Figure 2). In addition, Bifidobacterium relative abundance was also significantly lowered, registering a decrease of 6.18% when compared with the Control group (p < 0.05) ( Figure 2). The results are displayed as mean ± SEM. * indicates a significant difference (p < 0.05); "ns" indicates no significance (p > 0.05).

Effect of ZEN on the SCFA Colonic Content, pH Value and Ammonia Concentration
The SCFA concentrations among the experimental groups are presented in Table 2. The levels of acetic, propionic, isobutyric, butyric, isovaleric and valeric acids as well as total short-chained fatty acids did not significantly differ between the experimental groups for ZEN 75 or ZEN 290 when compared to the control. The highest SCFA concentrations were observed for acetic, propionic and butyric acid, irrespective of treatment. On the opposite side, isovaleric and isobutyric acids had the lowest observed values. The total concentration of SCFAs was slightly higher in the ZEN 290 group due to elevated concentrations of acetic acid, and propionic acid observed within this group but not significant.
ZEN presence within the diet did not alter in any significant way the pH values or ammonia concentrations between the experimental groups.

Effect of ZEN on the SCFA Colonic Content, pH Value and Ammonia Concentration
The SCFA concentrations among the experimental groups are presented in Table 2. The levels of acetic, propionic, isobutyric, butyric, isovaleric and valeric acids as well as total short-chained fatty acids did not significantly differ between the experimental groups for ZEN 75 or ZEN 290 when compared to the control. The highest SCFA concentrations were observed for acetic, propionic and butyric acid, irrespective of treatment. On the opposite side, isovaleric and isobutyric acids had the lowest observed values. The total concentration of SCFAs was slightly higher in the ZEN 290 group due to elevated concentrations of acetic acid, and propionic acid observed within this group but not significant.
ZEN presence within the diet did not alter in any significant way the pH values or ammonia concentrations between the experimental groups.

Correlations between SCFAs and Selected Microbial Populations
The effect of ZEN concentrations on the experimental groups was further expanded by Pearson's correlations between microbial species and SCFA production within the colon, presented in Table 3. The data were also represented in three heatmaps, corresponding to the Control group that received the standard diet and the ZEN dietary groups with differing ZEN concentrations ( Figure 3). Positive correlations pointed to a high probability of association, and a negative correlation was related to an absence of a relationship rather than an inhibitory process. The calculated correlations for the Control group between bacteria and SCFAs revealed that Lactobacillus was strongly associated with valeric acid (p = 0.0214) and moderately associated with isobutyric acid (p = 0.0399) within the colon (Table 3, Figure 3a). Additionally, the Clostridium genus was highly correlated with propionic acid (p = 0.0288) within the colon of piglets unaffected by ZEN. Several positive associations were observed for each microbial genus at different ZEN concentrations (Table 3, Figure 3). At 75 ppb ZEN concentration, both Lactobacillus (p = 0.0353) and Bifidobacterium (p = 0.018) were highly correlated with the presence of butyrates (Table 3, Figure 3b). Other strong positive correlations were observed between Bifidobacterium and isovalerates (p = 0.0189). Moderate correlations were observed between Enterobacter and both acetate (p = 0.0439) and propionate (p = 0.0346), as well as between Prevotella and propionates (p = 0.0156). The SCFA landscape changed at 290 ppb ZEN (Table 3, Figure 3c). Lactobacillus (p = 0.0155) and Bifidobacterium (p = 0.0244) maintained correlations with butyrates. Similarly, to its previous pattern, Enterobacter maintained a moderate correlation with acetates (p = 0.0144) and propionates (p = 0.0416). Strongly negative correlations were also observed between acetic acid, Bifidobacterium (p = 0.0124), and Lactobacillus (p = 0.0267). Additionally, Bifidobacterium was moderately negatively correlated with propionic acid (p = 0.0302) (Table 3, Figure 3c).

Effect of Zearalenone on Secretory IgA Synthesis
In order to evaluate the effect of ZEN on the secretory IgA (sIgA) contents in the intestinal mucosa, we have analysed the IgA levels in the intestinal content ( Figure 4). The results showed that exposure of piglets to 290 ppb ZEN can significantly increase the concentration of sIgA in the colonic content (1175 ng/mL versus 789 ng/m in the contro group). In contrast, no significant effect on sIgA concentration was observed for the ZEN 75 piglets group.

The Effect of Dietary Zearalenone on Tight Junction Proteins
The present study assessed the effect of ZEN at 75 ppb and 290 ppb concentrations on tight junction proteins at both gene and protein expression. No significant effect was

Effect of Zearalenone on Secretory IgA Synthesis
In order to evaluate the effect of ZEN on the secretory IgA (sIgA) contents in the intestinal mucosa, we have analysed the IgA levels in the intestinal content ( Figure 4). The results showed that exposure of piglets to 290 ppb ZEN can significantly increase the concentration of sIgA in the colonic content (1175 ng/mL versus 789 ng/m in the control group). In contrast, no significant effect on sIgA concentration was observed for the ZEN 75 piglets group.

Effect of Zearalenone on Secretory IgA Synthesis
In order to evaluate the effect of ZEN on the secretory IgA (sIgA) contents in intestinal mucosa, we have analysed the IgA levels in the intestinal content ( Figure 4). results showed that exposure of piglets to 290 ppb ZEN can significantly increase concentration of sIgA in the colonic content (1175 ng/mL versus 789 ng/m in the con group). In contrast, no significant effect on sIgA concentration was observed for the 75 piglets group.

The Effect of Dietary Zearalenone on Tight Junction Proteins
The present study assessed the effect of ZEN at 75 ppb and 290 ppb concentrations on tight junction proteins at both gene and protein expression. No significant effect was observed for the ZEN 75 group or ZEN 290. In the case of gene expression, a very slight increase was observed for ZO1 at 75 ppb ZEN and 290 ppb but without statistical significance ( Figure 5). Concerning protein expression, the western blot analysis for all the analysed marke validated the qPCR results, did not significallmt;ly affect with either of the two ZEN concentrations significantly affecting the junction proteins Cldn 4, ZO1 or Occldn (Figur 6).

Microbial Community Clustering and Relationship between
Clusterization of the selected microbial abundances data was checked by employin the Partial Least Squares discriminant analysis (PLS-DA) by regressing on many samp features (e.g., relative abundances) and finding the set of latent, orthogonal factors th help with cluster separation. It is often employed in -omics relative abundances, for ex ample, to assess the discriminatory ability of experimental groups associated with a di ease, toxin or phenotype. As shown in Figure 7, there was a clear distinction between ex perimental groups affected by ZEN and control in their microbiological composition. Th differences were more significant between the ZEN 290 group and the rest, this grou being the most distant from either the Control or ZEN 75 group. Concerning protein expression, the western blot analysis for all the analysed markers validated the qPCR results, did not significallmt;ly affect with either of the two ZEN concentrations significantly affecting the junction proteins Cldn 4, ZO1 or Occldn ( Figure 6). Concerning protein expression, the western blot analysis for all the analysed marke validated the qPCR results, did not significallmt;ly affect with either of the two ZE concentrations significantly affecting the junction proteins Cldn 4, ZO1 or Occldn (Figu 6).

Microbial Community Clustering and Relationship between
Clusterization of the selected microbial abundances data was checked by employin the Partial Least Squares discriminant analysis (PLS-DA) by regressing on many samp features (e.g., relative abundances) and finding the set of latent, orthogonal factors th help with cluster separation. It is often employed in -omics relative abundances, for e ample, to assess the discriminatory ability of experimental groups associated with a di ease, toxin or phenotype. As shown in Figure 7, there was a clear distinction between e perimental groups affected by ZEN and control in their microbiological composition. Th differences were more significant between the ZEN 290 group and the rest, this grou being the most distant from either the Control or ZEN 75 group.

Microbial Community Clustering and Relationship between
Clusterization of the selected microbial abundances data was checked by employing the Partial Least Squares discriminant analysis (PLS-DA) by regressing on many sample features (e.g., relative abundances) and finding the set of latent, orthogonal factors that help with cluster separation. It is often employed in -omics relative abundances, for example, to assess the discriminatory ability of experimental groups associated with a disease, toxin or phenotype. As shown in Figure 7, there was a clear distinction between experimental groups affected by ZEN and control in their microbiological composition. The differences were more significant between the ZEN 290 group and the rest, this group being the most distant from either the Control or ZEN 75 group. To investigate any other existing associations and to better visualize the data obtained, another PLS-DA analysis was performed on the following sets of parameters: SCFAs concentrations, microbial genera, pH value, and NH3 content with the frame of the experimental groups' vectors ( Figure 8). ZEN at 75 ppb was strongly associated with the isobutyric and isovaleric volatile acids. The pH, acetic acid concentration, and relative abundances of Clostridium and Enterobacter formed a cluster strongly associated with the presence of ZEN at 290 ppb and moderately associated with the propionic acid and ammonia concentrations.  To investigate any other existing associations and to better visualize the data obtained, another PLS-DA analysis was performed on the following sets of parameters: SCFAs concentrations, microbial genera, pH value, and NH 3 content with the frame of the experimental groups' vectors ( Figure 8). ZEN at 75 ppb was strongly associated with the isobutyric and isovaleric volatile acids. The pH, acetic acid concentration, and relative abundances of Clostridium and Enterobacter formed a cluster strongly associated with the presence of ZEN at 290 ppb and moderately associated with the propionic acid and ammonia concentrations. To investigate any other existing associations and to better visualize the data obtained, another PLS-DA analysis was performed on the following sets of parameters: SCFAs concentrations, microbial genera, pH value, and NH3 content with the frame of the experimental groups' vectors ( Figure 8). ZEN at 75 ppb was strongly associated with the isobutyric and isovaleric volatile acids. The pH, acetic acid concentration, and relative abundances of Clostridium and Enterobacter formed a cluster strongly associated with the presence of ZEN at 290 ppb and moderately associated with the propionic acid and ammonia concentrations.

Discussion
ZEN, a commonly found contaminant within animal feed, has been widely studied for its toxic effects in swine [2,3,[27][28][29]. Comparatively few studies have investigated the effect of ZEN at concentration of 0.100 mg ZEN/kg feed (the limit of EC recommendation) or lower in pig [27]. According to existing studies, the effects of piglets' exposure to low concentrations of ZEN, under 0.1 ppm, proved challenging to predict [30]. Time of exposure and the quantities of ingested feed are contributing factors that increase the prognosis difficulty. Therefore, even low concentrations of ZEN can induce unexpected responses; the host self-defence mechanisms might ignore the presence of mycotoxin, and the deleterious effects slowly accumulate [30,31]. It is also well known that the diet shift after weaning leading to a second change in bacterial populations. As such, feed quality must be given great importance [20,32]. For example, contaminants in feed, such as ZEN within the intestinal tract can alter microbial ecology homeostasis [7]. If mycotoxin contamination is associated with different pathogens infection as E. coli, Salmonella, and Rotaviruses (due to an immature, emerging immune system), this can lead to the apparition of dysbiosis, intestinal oxidative stress, inflammation, and diarrhoea [33,34].
In the present study, we investigated the effect of a low concentration of ZEN on microbial abundance and other associated markers such as SCFAs, IgA production, pH, ammonia content, and intestinal integrity through junction proteins in the colonic content and colon tissue of piglets fed a diet contaminated with 75 ppb of ZEN (level under the EC recommendation). These parameters were chosen to consider that gut microbiota plays an essential role in developing the immune and digestive systems facilitating nutrient absorption and preventing pathogen colonisation. It also binds ingested toxins and produces antimicrobial molecules and vitamins [35,36]. Within the intestinal tract, the microbiota improves the anti-inflammatory response as well as maintains the intestinal barrier function [36,37]. In addition, SCFAs, tight junction proteins and IgA production are crucial for maintaining intestinal barrier integrity, defence response and gastrointestinal health. In tandem and for comparison reasons, a higher level of ZEN, 290 ppb, above the EC recommendations was also analysed.
Our results showed that at 75 ppb (0.075 mg/kg feed) concentration, ZEN significantly (p < 0.05) reduced the Lactobacillus population within the colon, with the Bifidobacterium population lowered as well, but not in a significant way. A more severe alteration was registered for the 290 ppb (0.290 mg/kg feed) concentration, which decreased both Lactobacillus and Bifidobacterium significantly while noticeably (p < 0.05) increasing the populations of Prevotella, Clostridium, and Enterobacter genera. Although the Clostridium and Enterobacter genera include well-known pathogens, many of the members belonging to these genera are regular bacterial species representative of a healthy pig microbiota, as pointed out by many other studies [38][39][40]. Nevertheless, our findings show an increase in Clostridium and Enterobacter populations in the presence of ZEN 290 ppb. This was observed for Clostridium difficile in gilts by Cieplinska et al. [7] in challenges with ZEN ranging from 5 to 15 µg ZEN/kg b.w. Le Sciellour et al. [3] similarly reported an increase in Clostridium genera in the presence of both ZEN (0.75 mg/kg feed) and DON (3.02 mg/kg feed) in an acute seven-day challenge in female finishing pigs. In a study by Wang et al., [13] an increase in the Enterobacter population was reported, along with Escherichia, Bacteroides, and Salmonella, in the presence of ZEN. These opportunistic strains can lead to intestinal inflammations in piglets and generate substantial losses in commercial farms [41]. Despite evidence showing the capacity of Lactobacillus, along with other lactic acid bacteria such as Bifidobacterium, for mycotoxin absorption and biotransformation [12,42] certain studies observed that zearalenone could affect these populations, inducing a decline in their abundance. Li et al. 2018 [43] reported a noticeable decrease in the Lactobacillus population at a concentration of 400 µg ZEN/kg b.w. in rabbits in a 28 days challenge. Tan et al. [44] showed a 35% decline in Lactobacillus populations at a much lower concentration (40 µg ZEN/kg b.w.) along with a significant change in bacterial populations in mice. A reduction in Lactobacillus abundance was also observed in a study conducted on broilers by Jia et al., [4] this effect is correlated with their binding capacity. Moreover, Saenz et al. [45] observed a decrease in the whole taxonomic order of Lactobacillales in a 28-day ZEN (1623 ppb) challenge of piglets while observing an increase in the Bifidobacteriaceae. The Lactic Acid Bacteria complex, of which Lactobacillus and Bifidobacterium are members, usually plays a crucial role by producing many SCFAs important to the host and the microbial ecosystem [46]. Being a product of microbial fermentation of refractory carbohydrates, SCFAs are important for maintaining intestinal homeostasis, providing energy to colonocytes and preventing other intestinal diseases [47]. In piglets, for example, propionate is involved in the liver gluconeogenesis process, while acetate is a precursor in synthesising more complex fatty acids [48]. Of the SCFAs, butyrate is important in reducing intestinal inflammation and diminishing systemic infections, acting as an important bacterial and metabolic modulator [49,50]. The health of gastrointestinal tract tissue, mineral absorption and inflammation can also be influenced by normal concentrations of SCFAs [49]. In the present study, acetic acid was found in the most abundant quantity, followed by propionic acid, throughout all the experimental groups. As mentioned above, SCFAs are relevant for the microbial population in the gut. For instance, SCFAs can directly reduce the growth of Enterobacter by lowering the pH [25]. They also can reduce the colonisation of Enterobacter by suppressing inflammations [51].
These observations could explain why the decrease in Lactobacillus and Bifidobacterium populations by ZEN at 290 ppb would increase Enterobacter and Clostridium abundances [52]. In our study, ZEN had no significant effect on SCFAs production in the colon, either at 75 ppb or 290 ppb concentrations, when compared to Control. By contrast, other studies reported a decrease in SCFAs overall under the influence of ZEN [7,9,13,16] while Liu et al. observed an increase in SCFAs [53]. Although not significant, a slight increase in the ammonia values could be seen in the presence of both ZEN concentrations in the diet. Any difference in pH concentration was registered irrespective of the treatment. Because pH guides the competition between different microbial species and pH differs in different colon segments, it was necessary to investigate this parameter, comparing it to physiologically healthy pH ranges within the colon [54]. The values obtained ranged from 6.69 in the Control group to 6.73 and 6.79 in ZEN 75 and ZEN 290, respectively, considered to be within the physiologically normal range [55]. Bacterial metabolism cooperates in producing a pH gradient throughout the colon from the proximal to the distal section [56]. With the absorption of SCFAs within the colon, the pH increases with cells also secreting bicarbonate [57]. Another factor contributing to the increase in pH is the subsequent release of ammonia and urea in the distal colon through protein and amino acids metabolisation. This process increases the pH close to 6.6 [54,56]. Ammonia is a toxic metabolite within the gastrointestinal tract of pigs resulting from amino acid deamination or urea hydrolysis [58]. It readily passes into the portal blood, metabolised by the liver into urea that is then eliminated in urine [59]. Ammonia concentration rests on the equilibrium derived from amino acid deamination on the one hand and microbial protein synthesis on the other hand [58]. Usually, an ammonia increase leads to the inhibition of growth and differentiation of intestinal epithelial cells [60], and thus impacts the colon health negatively as well as disturbing the bacterial balance within piglets' gastrointestinal tract [61]. As mentioned above, no significant differences were observed in our study.
Furthermore, Pearson's correlations suggest different relationships between microbiota and SCFAs production under both ZEN concentrations. Butyric acid was strongly correlated with the presence of Lactobacillus and Bifidobacterium within the ZEN 75 experimental group. Other studies have also mentioned a similar association [62,63]. Enterobacter was found to be moderately associated with both acetic acid and propionic acid in the ZEN 75 and 290 experimental groups. This association was also observed in several other trials [64,65] under differing experimental premises.
The changes induced by ZEN on bacterial abundances were also verified by clustering, using two PLS-DA analyses. While the microbial taxonomic and relative abundance composition for the ZEN 75 group was similar to those of the Control, both differed from the ZEN 290 group. The high concentration of ZEN (290 ppb) profoundly affected the relative abundances and bacterial composition, with an apparent distancing between the experimental groups being highlighted. It was also strongly associated with an increased presence of Clostridium and Enterobacter and with the slight differences in concentrations of acetic acid within the colon content while negatively associated with the Lactobacillus genera. These associations could also contribute to the apparition of diarrhoea symptoms and contribute to it. Many studies discussed the relationship between Lactobacillus and post-weaning diarrhoea [66][67][68] showing that lactic acid bacteria are frequently used as a probiotic in reducing diarrhoea incidence, as reported by Dell'Anno et al. [69] or Wang et al. [70]. Other studies also indicate increased diarrhoea incidence with decreased Lactobacillus counts [71,72].
In the gut, the microbiota interacts with the host's immune system [73]. Secretory immunoglobulin A (sIgA) is the most abundant antibody isotype produced by B cells at mucosal surfaces as a response to the food antigens and the intestinal microbiota [74]. sIgA can neutralize toxins at the mucosal surfaces of the gut, protecting against pathogens and toxins [75]. In our experiment, the exposure of weaned piglets to zearalenone for 22 days induced a dose-dependent increase in sIgA in the colonic content, which was statistically significant in piglets exposed to the highest concentration of ZEN. Another recent study of Wang et al., [76] has similarly shown that the exposure to higher concentrations of ZEN (20 mg/kg b.w.), but for a shorter period (one week) can significantly induce an increase in faecal IgA levels. The sIgA synthesis is modulated by the complexity of gut microbiota and different bacterial populations can trigger an IgA response of different type and intensity [77]. On the other hand, sIgA can bind and coat the gut microorganisms controlling the microbiota composition, modulating the bacterial behaviours and enforcing host-microbiota homeostasis [78]. In the present study, the increase in sIgA could result in a defence response to the relative abundance of Prevotella, Clostridium and Enterobacter induced by ZEN. This could constitute a possible defence mechanism to regulate gut microbiota composition and to maintain gut homeostasis after toxin exposure by controlling the microorganisms' mechanisms, reducing their inflammatory potential and facilitating their clearance from the gut [75,79].
Within the intestinal barrier, tight junctions are essential in forming selectively permeable seals between adjacent intestinal epithelial cells [18]. These seals are composed of transmembrane proteins (Claudins), TJ-associated marvel proteins (e.g., Occludin), junctional adhesion molecules (JAMs), and cytosolic proteins (e.g., Zonula). ZEN can damage these seals, decreasing intestinal permeability [20,24]. No significant alterations in the gene expressions for Claudin 4, ZO1, or Occludin could be observed within the colon tissue in the present study. These results obtained by qPCR were confirmed at the protein level by Western blot analysis, showing that neither the concentration of ZEN below the EC recommendation (75 ppb) nor that above (290 ppb) had any effect on these junction proteins. Zhang et al. in 2021 [27] reported similar results when ZEN at 150 ppb did not induce changes in gene and protein expression of Claudin 1, Occludin, or ZO1 junction proteins in weaned piglets. By contrast, a significant decrease in the expression of these proteins was observed only at increased concentrations of 1500 to 3000 ppb ZEN [27], leading to cecal physical barrier injury.

Conclusions
Weaned piglets' exposure to contaminated feed with 75 ppb ZEN did not significantly impact the microbiota abundance or SCFA production within the colon, except for a significant decrease in Lactobacillus abundance. Nevertheless, this decrease could also be attributed to the microbiota changes when piglets change their diet after weaning. PLS-DA also shows that the differences between Control and ZEN 75 were not radical at a taxonomic level or relative abundance.
ZEN at 290 ppb also decreased the populations of Lactobacillus and Bifidobacterium significantly. In addition, it increased the relative abundance of Clostridium, Enterobacter and Prevotella compared to the Control. By contrast, there was a weak association between ZEN at 290 ppb and NH3 presence within the colon. PLS-DA analysis confirmed the existence of these changes at a microbiological level. ZEN 290 ppb also increased the percentage of diarrhoea incidence and IgA concentration (p < 0.05), while SCFA synthesis in the colon remained largely unaffected at both concentrations of ZEN.
As such, the presented findings bring new data regarding the effect of low concentrations of ZEN on young pigs. However, further analysis is needed to understand the underlying mechanisms that ZEN disrupts at the intestinal level.

Experimental Design
A total of 18 crossbred weaned piglets (TOPIGS-40) were used for the in vivo experiment and were randomly assigned to three groups (6 animals/group) as follows: (1) (Table 4). Piglets were individually ear-tagged, and every piglet was considered an experimental unit, with six experimental units per dietary group. Each group was housed in a separate pen. During the experimental trial, diarrhoea was recorded daily for each piglet. The number of days with diarrhoea was counted for each experimental group and expressed as a percentage of the total experimental period (22 days). Before ingesting the mycotoxin-contaminated diets, weaned piglets were acclimatized for one week. The young piglets were clinically normal, and no deaths resulted from the experiment. During the experiment, piglets had free access to solid feed and water. After 22 days, all animals (6 animals/group) were electrically stunned and exsanguinated (according to the 2010/63/EC directive of the EU Council). Colon and colonic content samples were collected from each animal and stored at −80 • C until further analyses. The handling and protection of the animals used in this experiment were realized in compliance with the 206/2004 Romanian law and the 98/58/EC Council Directive of the EU Council concerning treating animals used for experimental purposes.

Toxins
ZEN (>98% pure by high-performance liquid chromatography [HPLC]) used to contaminate the experimental diets was purchased from FERMENTEC (Jerusalem, Israel) and dissolved in DMSO (dimethyl sulfoxide): water (1:7) solution and then mixed into the basal diet in order to provide a feed containing 75 µg/kg 75 ppb and 290 µg/kg 290 ppb concentration confirmed by ELISA analysis. Experimental diets were screened by ELISA for other mycotoxins (aflatoxin B1, deoxynivalenol, ochratoxin A, fumonisins, and toxin T2) using ELISA kits Veratox (Neogen, Lansing, MI, USA) according to the manufacturer's instructions. Their concentrations were under the detection limits of the kits.

Microbial DNA Extraction
A QIAamp DNA stool minikit extraction kit (QIAGEN, Dusseldorf, Germany) was used for microbial genetic material extraction. A previous study already described primer sequences (Eurogentec, Seraing, Belgium) and the protocol of PCR amplification of universal 16S regions [80]. Standard curves were generated using serial dilutions of template DNA (105-1012 molecules/µL). The selective bacteria genera DNA was quantified using the RotorGene Q series PCR system (Qiagen, Hilden, Germany) with species-specific primers sequences designed for the following genera: Lactobacillus, Clostridium, Enterobacter, Prevotella and Bifidobacterium and using a PCR protocol already described elsewhere [80,81]. The gene copy numbers for the selected microorganisms were obtained using the Cq values relative to the standard curve. The final copy number for each targeted bacteria genus was calculated using the following equation: No. bacterial copies digesta/grams = (CM × AC × DV)/q × S in which CM represents the quantitative mean of the copy number, AC represents the DNA concentration of each sample, DV is the dilution volume of extracted DNA, q is the DNA amount (ng) which was used in the experiment, and S stands for the weight of the colonic sample (g) [62].

Determination of SCFAs, Ammonia Content and pH Value
A volume of 25 mL of digesta from the colon was used for pH analysis using a digital pH meter (Dostmann electronic, GmbH, Wertheim, Germany) following the manufacturer's protocol [82]. The ammonia concentration was measured using an ammonia selective-ion electrode (Orion 95-12, Orion Research Inc., Franklin, MA, USA) attached to a selective ion meter (710A model, Orion Research Inc., Franklin, MA, USA). The calibration was performed using dilutions at 10, 100, and 1000 mg/L of a 0.1 M standard ammonium chloride solution. In addition, an ISA solution (Ionic Strength Adjuster, Orion Research Inc., Franklin, MA, USA) of 0.5 mL was mixed into every 25 mL sample to ensure a uniform background ionic strength [83] The final results were reported as µmol/g. Short-chain fatty acids from the piglet colonic digesta samples were analysed in aqueous extracts by gas chromatography as described by Marin et al. [80]. Briefly, the aqueous extracts were injected into a gas chromatograph (Varian, 430-GC, Varian Inc., Palo Alto, CA, USA) after centrifugation. The GC instrument is fitted with an Elite-FFAP capillary column (inside diameter 320 mm) (Perkin Elmer, Waltham, MA, USA). Volatile fatty acids obtained from CRM46975, Supelco, Bellefonte, PA, USA, were used as standards. The final results were reported as µmol/g.

Quantification of Gene Expression by qPCR
The effects produced by the experimental diets on genes encoding for tight junction markers Claudin 4 (CLDN4), Zonula1 (ZO1), and Occludin (OCCLDN) were determined by qPCR. Methods cited in Pistol et al., 2021 were used to extract total ARN, the transcription into complementary DNA (cDNA) and the quantitative PCR [84]. The Excel-based NormFinder software was utilised to normalise qPCR data and reference genes selected from 6 housekeeping genes analysed. The 2(−∆∆C)q 2 (−∆∆Ct) formula was used for the comparison to the control group, thus obtaining the Fold Change.

Detection of Mucosal IgA in the Colonic Content
Lyophilised colonic content (0.25 g) was first diluted in 1 mL PBS and incubated at 40 • C for ten minutes, followed by a five minutes centrifugation at 5590G. Next, a 1:2 dilution in a solution of glycerol 40% in PBS was used for the supernatant. The IgA concentration in the samples was analysed using a pig IgA ELISA kit (Bethyl Laboratories Montgomery, TX, USA) according to the manufacturer's instructions. Next, the absorbance was read at 450 nm using a plate multi-reader (Varioscan, Thermo Scientific, Waltham, MA, USA), and the IgA content (ng/mL) was calculated.

Immunoblotting Analysis
Protein expression of three tight junction proteins: Claudin 4 (CLDN4), Zonula 1 (ZO1), and Occludin (OCCLDN), was determined by Western blot. The colon samples (50 mg) were homogenised in RIPA buffer. A Pierce BCA Protein Assay Kit, Thermo Fischer Scientific, USA, was used to quantify the protein content within the obtained lysates. A 10% SDS-PAGE electrophoresis was performed, and 30 µg of proteins were moved to the nitrocellulose membrane, blocked and incubated with specific primary and secondary antibodies as described by Pistol et al., 2021 [84]. The MicroChemi Imager (DNR Bio-Imaging Systems LTD, Neve Yamin, Israel) was used to develop the immunoblotting images, and the GelQuant software (DNR Bio-Imaging Systems LTD, Neve Yamin, Israel) was utilised to assay the protein expression level. A ratio of the expression levels between the protein of interest and β-actin was calculated to express the results.

Statistical Analyses
Results are presented as mean ± SEM. XLSTAT (Addinsoft, Paris, France) was used to perform a one-way ANOVA analysis and derive significance between the three experimental groups and Dennett's and Tukey's HSD tests. Statistical significance was considered for p values < 0.05 (* p < 0.05, ** p < 0.01, **** p < 0.0001). Partial least square-discriminant analysis was performed using XLSTAT (Addinsoft, Paris, France) to analyse clustering occurrence and the separation magnitude among the experimental groups and variables. Pearson's correlation coefficients between SCFA concentration and microbiota in the colon content as well as associated heatmaps, were calculated using XLSTAT (Addinsoft, Paris, France).  Data Availability Statement: The analysed sets of data used in the present paper can be offered, on reasonable request, by the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.